SK138893A3 - Method of the preparation of d-aminoacids or d-aminoacid derivatives - Google Patents

Abstract

The present invention relates to a new method for the preparation of D-aminoacids or derivatives of D-aminoacids from the racemic mixture of the corresponding hydantoin using cells of Agrobacterium radiobacter immobilized by inclusion into natural polymer gels. <IMAGE>

Description

Process for the preparation of D-amino acids or D-amino acid derivatives

Technical field

The invention relates to a novel process for the preparation of D-amino acids or D-amino acid derivatives from a racemic mixture of the respective hydantoin using Agrobacterium radiobacter cells immobilized by the addition of gelatinized natural polymers.

The use of biocatalysts based on immobilized microorganisms provides considerable advantages in continuous operation. The invention thus relates to the stereoselective biochemical transformation of D-hydantoin in order to obtain the corresponding D-amino acid. The products obtainable in this way are of interest to the pharmaceutical industry, such as p-hydroxyphenylglycine.

BACKGROUND OF THE INVENTION

The use of a hydantoinase and / or carbamylase enzyme system for transforming hydantoins is well known (Biotechnology: A Comprehensive Treatise Vol. 6a, H-J. Rehm, G. Reed Eds., Verlag Chemie, Weinheim, 1984).

A number of microorganisms have been proposed as producers of the above-mentioned enzyme system, inter alia. Pseudomonas, Agbacterium, Candida, Hansenula, Arthrobacter. Particular interest has been directed to Agrobacterium radiobacter, due to the fact that it provides an entire enzymatic system that exhibits both activities.

Of the patents related to this subject, it should be noted those relating to the use of Agrobacterium radiobacter to obtain D-phenylglycine or its derivatives (DE-OS 2 621 076; DE-OS 2 920 963) and also the following articles: R Olivieri et al.: Enzymatic Conversion of N-carbamyl-D-amino acids to D-amino acids Enzyme Microb. Technol. 1, 201-204 (1979) and R. Olivieri et al., Microbial Transformation of Racemic Hydantoins to D-amino acids Biotechnol. Bioeng. 23, 2173-2183 (1981). These articles describe a microbial process using free biomass.

Other microorganisms providing enzyme systems include Agrobacterium tumefaciens (EP 0 309 310), Pseudomonas (DE-OS 3 018 581), Candida (JP 61 177 992), Hansenula (JP 61 177 991).

The use of immobilized microorganisms provides substantial operational advantages both in the manufacturing and separation processes of the present invention. Although the use of Agrobacterium radiobacter cells immobilized in gelatinized polymers has already been patented (ES 523 360), this biocatalyst has been used for agricultural purposes and the maintenance of the enzyme activity of hydantoinase and carbamylase has been neglected.

On the other hand, the use of other immobilized microorganisms for the bioconversion of hydantoins has been described, as in the case of EP 0 Bacillus brevis. Next 4 094 741. Claims of this

No. 261,836, relating to immobilization, U.S. Pat. of the patent include generally immobilizing the enzymatic activity of a number of microorganisms. However, there is no specific mention of Agrobacterium radiobacter or specific methods of immobilization.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides a novel process for the preparation of D-amino acids and D-amino acid derivatives from a racemic mixture of the corresponding hydantoin using Agrobacterium radiobacter cells which are immobilized by the addition of gelatinized natural polymers.

The use of immobilized microorganisms allows reuse of the biocatalyst. This results in substantial advantages, both in terms of separation and in terms of operation of the continuous process.

The use of Agrobacterium radiobacter cells has the advantage of providing the complete enzymatic system required for the quantitative transformation of D-hydantoin to the desired product.

The stereospecificity of the enzymes that act on the D-form of the hydantoin is based on disrupting the D-L-equilibrium of the racemic mixture, allowing the quantitative conversion of such a racemic mixture to the D-amino acid or D-amino acid derivative.

Immobilization of Agrobacterium radiobacter cells (which cells are intact or broken) is accomplished by the addition of gelatinous alginate or carageenate formed in the presence of calcium ions and / or generated by heat treatment. The spherical particles of controllable size thus obtained can optionally be further strengthened to increase their mechanical resistance.

As a result, a stable biocatalyst is obtained which is capable of fully converting D-L-hydantoin to the corresponding D-amino acid derivative, wherein the biocatalyst can be used for at least 22 days.

The invention is illustrated by the following example. This example is for illustrative purposes only and in no way limits the scope of the invention. The example is illustrated by the figures below.

An overview of FIG. in the drawings

In FIG. 1 depicts the pH dependence of hydantoinase activity.

In FIG. 2 shows the pH dependence of carbamylase activity.

In FIG. Figures 3 and 4 show temperature dependence of enzymatic activities.

In FIG. Figures 5 and 6 show the stability of free biomass at 45 and 50 ° C.

In FIG. 7 shows the stability of two enzymatic activities tested over 22 days at 40 ° C.

In FIG. 8 shows the performance of free biomass tested under the same conditions.

Demonstration examples

1. Agrobacterium radiobacter cells are used to obtain D-p-hydroxyphenylglycine from the corresponding hydantoin. The resulting product is obtained according to the following scheme.

HO

H — C-nh hydantoinase ho-c-ooh o = C C = o

NH m-C-nhII ^of o

D, L-hydantoin carbamylase

HO CH-COCW

NH 2

D-p-hvdroxyfenylglycín

2. Activity tests

2.1. The enzymatic activity of the Agrobacterium radiobacter biomass is determined as follows: 1 ml of the biocatalyst suspension is added to 11 ml of the hydantoin suspension in pH 8 buffer, with a final substrate concentration of 0.02M. The mixture was incubated for 20 minutes at 40 ° C under a nitrogen atmosphere and the obtained D-p-hydroxyphenylglycine was analyzed by high performance liquid chromatography (HPLC).

The same activity assays are used to determine carbamylase activity except that carbamylderivative is used instead of hydantoin.

The activity of the immobilized biocatalyst is determined in the same manner, with 5 ml of biocatalyst.

2.2 To determine the best pH for each enzymatic activity, 10 mM solutions of each of the substrates (hydantoin and carbamyl derivative) in tris-HCl buffer or carbonate / carbonic acid buffer were prepared, depending on the desired pH. A suspension of 1 ml of biomass is added to a 10 ml substrate solution. Testing shall be carried out at 40 ° C as described in paragraph 2.1.

2.3 To determine the best temperature for each enzymatic activity, substrates (10mM) were dissolved in tris-HCl buffer at the optimum pH for each case (pH 8 for hydantoinase activity and pH 8.5 for carbamylase activity). Activity assays are performed at various temperatures.

2.4 Hydantoinase activity is expressed in mmol / l of converted product over a 20 minute reaction (sum of product + carbamyl derivative). The carbamylase activity is expressed in mmol / L of p-hydroxyphenyl-glycine obtained after reaction for 20 minutes from the intermediate product.

3. Immobilization

Agrobacterium radiobacter cell suspension is immobilized in gelatinized alginate using the following procedure:

Materials: sodium alginate (Protanal LF 10/60) at 40% final volume. Agrobacterium radiobacter biomass is used in a concentration of 21% of the final volume, the concentration of calcium chloride is 1.5%.

ml of biomass (0.7 g / ml) was mixed with sodium alginate solution (2.6 g, 46.67 ml). The mixture is agitated magnetically until complete homogenization. Using a peristaltic pump, the gel is introduced into a hollow needle (0.5 mm diameter) located 30 cm from the mechanically shaken calcium chloride solution. Contact of the gel with calcium ions causes the droplets to solidify. All immobilization procedures are carried out under a nitrogen atmosphere.

4. Stability

4.1. The stability of free biomass ml of biomass shall be maintained at the test temperature and under a nitrogen atmosphere throughout the test. One milliliter samples are taken at predetermined time intervals and tested for activity.

2.4 Stability of immobilized biomass

Activity tests are periodically performed to determine the stability of the biocatalyst produced. The immobilized biocatalyst is stored in sealed containers at the desired temperature under a nitrogen atmosphere, the hydantoin concentration being 30 g / l. The biocatalyst is subjected to an overall washing procedure before conducting activity tests.

5. Results

1.5 Determination of working conditions for each enzyme activity

The pH dependence of the hydantoinase activity is shown in FIG. 1. A greater deviation is achieved at pH 8. Similarly, FIG. 8 shows the dependence of carbamylase activity on pH, and it is apparent that the optimum pH in this case is 8.5.

Fig. Figures 3 and 4 show the dependence of enzyme activity on temperature. Maximum hydantoinase activity is achieved at 45 ° C and maximum carbamylase activity is achieved at 50 ° C.

The stability of the free biomass at 45 and 50 ° C is apparent from FIG. Both enzymatic activities show high stability over 10 days, although the carbamylase activity at 50 ° C decreases by approximately 20%.

The data obtained in the activity and stability tests allow to conclude that the working temperature should be in the range of 45 to 50 ° C and the optimum pH in the range of 8 to 8.5. However, if less carbamylase activity is present in biomass, compared to hydantoinase activity, optimal values for carbamylase activity should be made in close proximity to: pH = 8.5 and temperature = 50 ° C.

2.5 Operation using immobilized biocatalyst.

The results obtained with the immobilized biocatalyst show that an activity retention of 50-80% is achieved (it should be taken into account that measurement of activity reflects not only the enzymatic activity itself but also the various limitations that occur with the immobilized biocatalyst) .

The biocatalyst particles obtained exhibit 100% stability with respect to the two enzymatic activities tested over a period of 22 days at 40 ° C (see Fig. 7).

The performance of the immobilized biocatalyst is the same as that of free biomass when tested under the same conditions (see Figure 8).

The immobilized biocatalyst is therefore able to maintain the enzymatic activities of Agrobacterium radiobacter cells under operating conditions and to perform a specific transformation of D-hydantoin to D-p-hydroxyphenylglycine.

Claims (5)

PATENT CLAIMS Process for the preparation of D-amino acids or D-amino acid derivatives from the corresponding hydantoin, characterized in that immobilized microorganisms containing hydantoinase and carbamylase enzymatic activity are used in the production. Method according to claim 1, characterized in that the microorganisms are immobilized by the addition of gelatinised natural polymers, for example alginate or carageenate. 3. The method of claim 2, wherein the microorganism is Agrobacterium radiobacter. 4. The process of claim 3 wherein the product obtained from the racemic hydantoin mixture is D-p-hydroxyphenylglycine. A process according to any one of claims 1, 2, 3 or 4, characterized in that it is carried out in a reducing environment, either in a nitrogen atmosphere or in the presence of chemical reducing agents, such as e.g. sodium sulfide.